Alfalfa variety 14XXP21R

ABSTRACT

A novel alfalfa variety designated 14XXP21R and seed, plants and plant parts thereof are provided. Methods for producing an alfalfa plant comprise crossing alfalfa variety 14XXP21R with another alfalfa plant. Methods for producing an alfalfa plant containing in its genetic material one or more traits transgenes or locus conversions introgressed into 14XXP21R through backcross conversion and/or transformation are provided and the alfalfa seed, plant and plant part produced thereby. Alfalfa seed, plants or plant parts produced by crossing alfalfa variety 14XXP21R or a locus or trait conversion of 14XXP21R with another alfalfa plant or population are disclosed. Alfalfa populations derived from alfalfa variety 14XXP21R, methods for producing other alfalfa populations derived from alfalfa variety 14XXP21R and the alfalfa populations and their parts derived by the use of those methods.

FIELD OF INVENTION

This invention is in the field of alfalfa (Medicago sativa) breeding,specifically relating to an alfalfa variety designated 14XXP21R.

BACKGROUND OF THE INVENTION

Alfalfa (Medicago sativa L., also known as lucerne) is one of theworld's most valuable forage legumes. It is grown for hay, pasture andsilage, and is valued highly as a livestock feed. Alfalfa is highlyeffective in nitrogen fixation and is frequently planted in croprotation to replenish nutrients depleted from the soil by other cropssuch as corn.

Alfalfa originated in the Near East, in the area extending from Turkeyto Iran and north into the Caucasus. From the great diversity of formswithin the genus Medicago, two species, M. sativa and M. falcate, havebecome important forage plants. These species are mainly tetraploid,with 32 chromosomes, although diploid forms are known.

The commercial production of seeds for growing alfalfa plants normallyinvolves four stages, the production of breeder, foundation, certifiedand registered seeds. Breeder seed is the initial increase of seed ofthe strain which is developed by the breeder and from which foundationseed is derived. Foundation seed is the second generation of seedincrease and from which certified seed is derived. Certified seeds areused in commercial crop production and are produced from foundation orcertified seed. Foundation seed normally is distributed by growers orseedsmen as planting stock for the production of certified seed.

SUMMARY OF THE INVENTION

Provided is a novel alfalfa variety, designated 14XXP21R and processesfor making and using 14XXP21R. Seed of alfalfa variety 14XXP21R, plantsof alfalfa variety 14XXP21R, plant parts of alfalfa variety 14XXP21R,and processes for making and using an alfalfa plant are provided. Theplant part may comprise at least one cell of alfalfa variety 14XXP21R ormodified as described herein. Methods of breeding that comprise crossingalfalfa variety 14XXP21R with another alfalfa plant are described. Inone aspect, processes for making an alfalfa plant containing in itsgenetic material one or more traits introgressed into 14XXP21R throughbackcross conversion and/or transformation, and to the alfalfa seed,plant and plant part produced by such introgression are provided. Plantcells and plants, seeds and plant parts comprising at least one cell ofalfalfa variety 14XXP21R or a locus conversion of variety 14XXP21R areprovided. Alfalfa seeds, plants or plant parts produced by crossing thealfalfa variety 14XXP21R or an introgressed trait conversion of 14XXP21Rwith another alfalfa population or variety. Alfalfa populations derivedfrom alfalfa variety 14XXP21R and processes for making other alfalfapopulations derived from alfalfa variety 14XXP21R are provided as wellas the alfalfa populations and their parts derived by the use of thoseprocesses.

DETAILED DESCRIPTION OF THE INVENTION

Alfalfa is a herbaceous perennial legume characterized by a deep taproot showing varying degrees of branching. Erect or semi-erect stemsbear an abundance of leaves. The number of stems arising from a singlewoody crown may vary from just a few to fifty or more. New stems developwhen older ones mature or have been cut or grazed. Flowers are borne onaxillary racemes which vary greatly in size and number of flowers.Flower color is predominantly purple, or bluish-purple, but other colorsoccur. The fruit is a legume, or pod, usually spirally coiled in M.sativa. Seeds are small and the color varies from yellow to brown.Alfalfa is widely adapted to temperature and soil conditions, except forhumid tropical conditions. Reproduction in alfalfa is mainly bycross-fertilization, but substantial self-pollination may also occur.Cross-pollination is effected largely by bees.

The following terms are used in this application:

Acid-Detergent Fiber (“ADF”) approximates the amount of cellulose fiberand ash present in a feed. Forages with high ADF values are lessdigestible than forages with low ADF values and, therefore, providefewer nutrients to the animal through digestion. Because of thisrelationship, ADF serves as an estimate of digestibility and can be usedby nutritionists to predict the energy that will be available from aforage.

AOSCA. Abbreviation for Association of Official Seed CertifyingAgencies.

Crude Protein (“CP”) is determined by measuring the total nitrogenconcentration of a forage and multiplying it by 6.25. This techniquemeasures not only the nitrogen present in true proteins, but also thatpresent in non-protein forms such as ammonia, urea and nitrate. Becausemost of the non-protein forms of nitrogen are converted to true proteinby the rumen microorganisms, CP is considered by nutritionists toprovide an accurate measure of the protein that will be available toruminant animals from a given forage.

DM. Abbreviation for Dietary Dry Matter. Used to calculate yield.

Fall Dormancy (Dormancy or “FD”) Most alfalfa plants go dormant in thefall in preparation for winter. The onset of dormancy is triggered by acombination of day length and temperature and is genotype dependent.Fall dormancy scores indicate the dormancy response of alfalfa genotypesby quantifying the height of alfalfa measured in October relative to aset of standard check varieties. The standard fall dormancy testrequires that plants are cut off in early September with plant heightmeasured in early-mid October. Early fall dormant types show very littlegrowth after the September clipping, later fall dormant type demonstratesubstantial growth.

Alfalfa is classified into fall dormancy groups, numbered 1 to 11, whereDormancy Group 1 is most dormant and suited for cold climates (suchvarieties would stop growing and go dormant over winter), and DormancyGroup 7-11 are very non-dormant and suited for very hot climates (suchvarieties would have high growth rates over a very long growing seasonand would have relatively high winter activity). The NA&MLVRB recognizesstandard or check varieties for Dormancy Groups 1-11, Check cultivarsare listed in the NAAIC Standard Tests to Characterize AlfalfaCultivars, maintained online on the NAAIC's website. The check varietiesfor the various fall dormancy ratings/Dormancy Groups (corresponding tothe rating scale used by the Certified Alfalfa Seed Council (CASC)) areas follows:

Check Cultivars: A single set of check cultivars representing falldormancy classes (FDC) 1 to 11 are designated. These check cultivarshave been selected to maintain the intended relationship between theoriginal set of nine check cultivars (Standard Tests, March 1991,updated in 1998) and to have minimal variation across environments. Theactual fall dormancy rating (FDR) based on the average University ofCalifornia regression and the Certified Alfalfa Seed Council Class thateach check cultivar represents are listed below.

Variety FDR FDC Maverick  0.8  1.0 Vernal  2.0  2.0 5246  3.4  3.0Legend  3.8  4.0 Archer  5.3  5.0 ABI 700  6.3  6.0 Doña Ana  6.7  7.0Pierce  7.8  8.0 CUF 101  8.9  9.0 UC-1887  9.9 10.0 UC-1465 11.2 11.0

Fall dormancy regression (FDR) number corresponds to the fall dormancyvalue calculated using the University of California regression equation.

Fall dormancy class (FDC) number corresponds to the fall dormancy classused by the Certified Alfalfa Seed Council (CASC)

In Vitro True Digestibility (“IVTD”) is a measurement of digestibilityutilizing actual rumen microorganisms. Although ADF serves as a goodestimate of digestibility, IVID provides a more accurate assessment of aforage's feeding value by actually measuring the portion of a foragethat is digested. This process is more expensive and time consuming thanthe analysis for ADF concentrations of a feed, but provides a moremeaningful measure of forage digestibility. Techniques for measuring invitro digestibility are based on incubating a forage sample in asolution containing rumen microorganisms for an extended period of time(usually 48 hours).

Milk Per Ton is an estimate of the milk production that could besupported by a given forage when fed as part of a total mixed ration.The equation for calculating milk per ton uses NDF and ADF to calculatetotal energy intake possible from the forage. After subtracting theamount of energy required for daily maintenance of the cow, the quantityof milk that could be produced from the remaining energy is calculated.The ratio of milk produced to forage consumed is then reported in theunits of pounds of milk produced per ton of forage consumed. Milk perton is useful because it characterizes forage quality in two terms thata dairy farmer is familiar with: pounds of milk and tons of forage. Bycombining milk per ton and dry matter yield per acre, we arrive at “milkper acre”. This term is widely used to estimate the economic value of aforage.

NAAIC. North America Alfalfa Improvement Conference, which is thegoverning body over the NA&MLVRB

NA&MLVRB. National Alfalfa and Miscellaneous Legume Variety ReviewBoard. The NA&MLVRB is administered by the Association of Official SeedCertifying Agencies (AOSCA).

NAVRB. Abbreviation for National Alfalfa Variety Review Board. NAVRBrecently changed its name to “National Alfalfa and Miscellaneous LegumeVariety Review Board” (NA&MLVRB).

Neutral-Detergent Fiber (“NDF”) represents the total amount of fiberpresent in the alfalfa. Because fiber is the portion of the plant mostslowly digested in the rumen, it is this fraction that fills the rumenand becomes a limit to the amount of feed an animal can consume. Thehigher the NDF concentration of a forage, the slower the rumen willempty reducing what an animal will be able to consume. For this reason,NDF is used by nutritionists as an estimate of the quantity of foragethat an animal will be able to consume. Forages with high NDF levels canlimit intake to the point that an animal is unable to consume enoughfeed to meet their energy and protein requirements.

Potato leafhopper (PLH) resistant variety. Potato Leafhopper Resistanceis a reaction of the alfalfa host plant which enables it to avoidserious damage from potato leafhopper feeding. The resistant plantreaction is to demonstrate normal growth in the presence of highpopulations of potato leafhoppers, whereas susceptible plants showsignificant stunting and yellowing in reaction to insect feeding. Theconvention used for measuring PLH damage disclosed herein is patternedafter standard tests used for measuring damage/resistance to otherpests. Individual plants are scored on a (1-5) scale, where 1=no damageevident and 5=severe stunting and yellowing. Plants scored as 1 and 2are classified as resistant. The average severity index (ASI) of avariety is the average damage score for 100 random plants. The ASI isoften used in combination with percent resistance to characterize pestresistance of alfalfa cultivars. Using this standard convention, analfalfa variety described as being resistant to PLH has between(31%-50%) of the plants in the variety being scored 1 or 2 in a standardtest to measure PLH reaction. Individual alfalfa plants or clones(clonal propagules of individual genotypes) with a resistance score of 1have very high resistance; a score of 3 show moderate resistance; and ascore of 5 show no resistance.

Relative Feed Value (“RFV”) is a numeric value assigned to forages basedupon their ADF and NDF values. In this calculation, NDF is used toestimate the dry matter intake expected for a given forage, and the ADFconcentration is used to estimate the digestibility of the forage. Bycombining these two relationships, an estimate of digestible dry matterintake is generated. This value is then reported relative to a standardforage (fall bloom alfalfa=100) and can be used to rank forages based ontheir anticipated feeding value. Relative feed value has been acceptedin many areas as a means of estimating forage feeding value and iscommonly used in determining the price of alfalfa at tested hayauctions.

Relative Forage Quality (“RFQ”) is a numeric value that estimates theenergy content of forage for total digestible nutrients as recommendedby the National Research Council. Values are assigned to forages basedupon the actual fiber digestibility (NDFd) and Total DigestibleNutrients (TDN). By combining these two relationships, an estimate ofhow the forage will perform in animal rations is predicted. Relativeforage quality has been accepted in many areas as a means of estimatingforage feeding value and is commonly used in determining the price ofalfalfa at tested hay auctions or for on farm use.

Synthetic variety (“SYN”) is developed by intercrossing a number ofgenotypes with specific favorable characteristics and/or overall generalfavorable qualities. Synthetic (SYN) variety can be developed by usingclones, inbreds, open pollinated varieties, and/or individualheterozygous plants.

TA. Tons per Acre. Used to calculate yield.

Total Digestible Nutrients (“TDN”) is an estimate of the energy contentof a feedstuff based on its relative proportions of fiber, fat,carbohydrate, crude protein, and ash. Because it is expensive to measureeach of these components, TDN is usually estimated from ADF or IVTD.Although still used in some areas as a criteria for evaluating alfalfahay at auctions, TDN has been shown to overestimate the energy contentof low quality forages and thus does not accurately reflect thenutritional value of all forage samples.

Winterhardiness (“WH”) is a measure of the ability of an alfalfa plantto survive the stresses associated with winter. Cold hardiness is a keyfeature of the winterhardiness trait. There is a general relationshipbetween fall dormancy and winterhardiness, the early fall dormant types(FD2-5) being more winterhardy than the later fall dormant types(FD6-9). The winterhardiness rating used in this patent are derived fromthe standard test for measuring winter survival. The standard testmeasures plant survival and spring vigor following a winter stressenough to substantially injure check varieties.

Alfalfa varieties are heterogeneous populations formed by intercrossinga number of alfalfa clones. Pest resistance in alfalfa varieties iscommonly measured in standard tests as the percent of plants in thepopulation that express the resistance trait. The National AlfalfaVariety Review Board in accordance with the recommendation of the NorthAmerican Alfalfa Improvement Conference has adopted a convention thatuses percent resistant plants to describe levels of pest resistance.This convention is as follows: (0-5%)=susceptible, (6-15%)=lowresistance, (16-30%)=moderate resistance, (31-50%)=resistance, and(>51%)=high resistance. With most pests, economic losses due to pestdamage are minimized or eliminated with varieties containing resistanceto high resistance. Individual plants can also have varying levels ofresistance.

Alfalfa is an auto-tetraploid and is frequently self-incompatible inbreeding. When selfed, little or no seed is produced, or the seed maynot germinate, or when it does, may have reduced vigor and may laterstop growing. Typically, fewer than five percent of selfed crossesproduce seed. When a very small population is crossbred, inbreedingdepression occurs, and traits of interest, such as quality, yield, andresistance to a large number of pests (e.g., seven or eight differentpests), are lost. Thus, producing a true breeding parent for hybrids isnot possible, which complicates breeding substantially.

Efforts to develop alfalfa varieties having improved traits andincreased production have focused on breeding for disease, insect, ornematode resistance, persistence, adaptation to specific environments,increased yield, and improved quality. Breeders have had some success inbreeding for increased herbage quality and forage yield, although thereare significant challenges.

Breeding programs typically emphasize maximizing heterogeneity of agiven alfalfa variety to improve yield and stability. However, thisgenerally results in wide variations in characteristics such asflowering dates, flowering frequency, development rate, growth rate,fall dormancy and winter hardiness. Prior art breeding methods do notemphasize improving the uniformity of these characteristics.

Some sources indicate that there are nine major germplasm sources ofalfalfa: M. falcata, Ladak, M. varia, Turkistan, Flemish, Chilean,Peruvian, Indian, and African. Tissue culture of explant source tissue,such as mature cotyledons and hypocotyls, demonstrates the regenerationfrequency of genotypes in most cultivars is only about 10 percent.Seitz-Kris, M. H. and E. T. Bingham, In vitro Cellular and DevelopmentalBiology 24 (10):1047-1052 (1988). Efforts have been underway to improveregeneration of alfalfa plants from callus tissue. E. T. Bingham, et.al., Crop Science 15:719-721 (1975).

Disclosed herein are methods for producing first-generation syntheticvariety alfalfa seed comprising crossing a first parent alfalfa plantwith a second parent alfalfa plant and harvesting resultantfirst-generation (F1) alfalfa seed, wherein said first or second parentalfalfa plant is one of the alfalfa plants of the present inventiondescribed above.

Alfalfa having agronomically desirable traits and breeding methods thatresult in a high degree of hybridity, uniformity of selected traits, andacceptable seed yields are described herein.

Methods of obtaining alfalfa populations using cytoplasmic male sterilealfalfa populations (A populations), maintainer alfalfa populations (Bpopulations), and male fertile pollenizer populations (C populations)are provided.

Male sterile A populations may be identified by evaluating pollenproduction using the Pollen Production Index (P.P.I.), which recognizesfour distinct classes: 1. Male Sterile Plants (MS) PPI=0 for which novisible pollen can be observed with the naked eye when flower is trippedwith a black knife blade; 2. Partial Male Sterile Plant (PMS) PPI=0.1for which a trace of pollen is found with the naked eye when flower istripped with a black knife blade; 3. Partial Fertile Plant (PF) PPI=0.6for which less than a normal amount of pollen can be observed with thenaked eye when flower is tripped with a black knife blade; and 4.Fertile Plant (F) PPI=1.0 for which normal amounts of pollen can beobserved when flower is tripped with a black knife blade.

The cells of the cytoplasmic male sterile (A population) alfalfa plantscontain sterile cytoplasm and the non-restorer gene. The maintainerpopulation (B population) is a male and female fertile plant, and whencrossed with an A population plant, maintains the male sterility of thecytoplasmic male sterile plant in the progeny. The cells of a maintainerpopulation plant contain normal cytoplasm and the non-restorer gene.Methods for identifying cytoplasmic male sterile and maintainerpopulations of alfalfa are well known to those versed in the art ofalfalfa plant breeding (e.g., see U.S. Pat. No. 3,570,181, which isincorporated by reference herein). A pollenizer population (Cpopulation) is a fertile plant containing both male and female parts.

Cytoplasmic male sterile populations may be maintained by vegetativecuttings. Maintainer populations can be maintained by cuttings orself-pollination. Male sterile plants can be obtained bycross-pollinating cytoplasmic male sterile plants with maintainerplants. Pollenizer populations can be maintained by selfing or, if morethan two clones are used, by cross-pollination.

At least one of the alfalfa plant populations used in developing alfalfaplants according to the methods described herein may have at least onedesirable agronomic trait, which may include, for example, resistance todisease or insects, cold tolerance, increased persistence, greaterforage yield or seed yield, improved forage quality, uniformity ofgrowth rate, and uniformity of time of maturity.

In the controlled pollination step, the cytoplasmic male sterile plantsare typically grown in separate rows from the maintainer plants. Theplants are pollinated by pollen-carrying insects, such as bees.Segregating the male sterile and maintainer plants facilitates selectiveharvest of seed from the cytoplasmic male sterile plants. The malesterile seed and male fertile seed can be provided as a random mixtureof the seed in a ratio of about 4:1, which would provide for randomdistribution of the male sterile and male fertile plants grown therefromand random pollination of the alfalfa plants. As one of skill in the artwill appreciate, one could also practice the method of the inventionusing designed distribution of male sterile and male fertile populationswithin a field and subsequent pollination by pollen-carrying insects.

One of ordinary skill in the art will appreciate that any suitable malesterile population, maintainer population, and pollenizer populationcould be successfully employed in the practice of the method of theinvention.

In an embodiment, a tissue culture of regenerable cells derived, inwhole or in part, from an alfalfa plant of synthetic variety named14XXP21R is provided. In one embodiment, cells may be regenerated intoplants having substantially all the morphological and physiologicalcharacteristics of the synthetic alfalfa variety named 14XXP21R that aredescribed in the attached tables. Some embodiments include a tissueculture that includes cultured cells derived, in whole or in part, froma plant part selected from the group consisting of leaves, roots, roottips, root hairs, anthers, pistils, stamens, pollen, ovules, flowers,seeds, embryos, stems, buds, cotyledons, hypocotyls, cells andprotoplasts. Another embodiment is an alfalfa plant regenerated fromsuch a tissue culture, having all the morphological and physiologicalcharacteristics of synthetic alfalfa variety 14XXP21R.

Tissue culture of alfalfa is further described in Saunders, J. W. andBingham, E. T., (1971) Production of alfalfa plants from callus tissue,Crop Sci 12;804-808, and incorporated herein by reference. Methods forregeneration of alfalfa plants from tissue culture are described in U.S.Pat. No. 5,324,646 issued Jun. 28, 1994, which is hereby incorporated byreference. Additionally, methods for improving heritable somaticembryogenesis in alfalfa, which may be controlled by relatively fewgenes, are provided, for example, methods of isolation of the geneticcontrol of embryogenesis and breeding methods which would incorporatesuch information.

A plant may include plant cells, plant protoplasts, plant cells oftissue culture from which alfalfa plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as pollen, flowers, seeds, leaves, roots, stems, and thelike.

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions, such as encoding specific protein products. DNA sequences,whether from a different species or from the same species, which areinserted into the genome using transformation are referred to hereincollectively as “transgenes”. Provided are methods of modifying alfalfavariety 14XXP21R by genome editing and locus conversions of alfalfavariety 14XXP21R produced by editing the genome of alfalfa variety14XXP21R. In some embodiments of the invention, a transformed or editedvariant of 14XXP21R may contain at least one transgene and/or gene editbut could contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or no morethan 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 transgenes and/orgene edits. Methods for producing transgenic and edited plants and theirused to create transformed and edited versions of alfalfa variety14XXP21R are provided.

Provided are plants, seeds and plant parts of alfalfa variety 14XXP21Rfurther comprising a locus conversion, and method for making and usingsuch plants, seeds and plant parts. A locus conversion, also called atrait conversion, can be a native trait, an edited trait, or atransgenic trait. In addition, a recombination site itself, such as anFRT site, Lox site or other site specific integration site, may beinserted by backcrossing and utilized for direct insertion of one ormore genes of interest into a specific plant variety. The trait ofinterest is transferred from the donor parent to the recurrent parent.

A single locus may contain several transgenes or edits, such as atransgene for disease resistance that, in the same expression vector,also contains a transgene for herbicide tolerance or resistance. Thegene for herbicide tolerance or resistance may be used as a selectablemarker and/or as a phenotypic trait. A single locus conversion of a sitespecific integration system allows for the integration of multiple genesat a known recombination site in the genome. At least one, at least twoor at least three and less than ten, less than nine, less than eight,less than seven, less than six, less than five or less than four locusconversions may be introduced into the plant by backcrossing,introgression or transformation to express the desired trait, while theplant, or a plant grown from the seed, plant part or plant cell,otherwise retains the phenotypic characteristics of the deposited seedwhen grown under the same environmental conditions.

The modified variety 14XXP21R or variety 14XXP21R further comprising alocus conversion may be further characterized as having all of, the sameor essentially all of or essentially the same phenotypic characteristicsor physiological and morphological characteristics of alfalfa variety14XXP21R, for example, as are listed in one or more of the tablesherein, when grown under the same or similar environmental conditionsand/or may be characterized by percent identity to 14XXP21R asdetermined by molecular markers, such as SSR markers or SNP markers.Examples of percent identity determined using markers include at least95%, 96%, 97%, 98%, 99% or 99.5%. Traits can be used by those ofordinary skill in the art to characterize the plants disclosed herein.Traits are commonly evaluated at a significance level, such as a 1%, 5%or 10% significance level, when measured in plants grown in the sameenvironmental conditions.

The backcross or locus conversion may result from either the transfer ofa dominant allele or a recessive allele. Selection of progeny containingthe trait of interest can be accomplished by direct selection for atrait associated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele, such as the waxy starchcharacteristic, requires growing and selfing the first backcrossgeneration to determine which plants carry the recessive alleles.Recessive traits may require additional progeny testing in successivebackcross generations to determine the presence of the locus ofinterest. The last backcross generation is usually selfed to give purebreeding progeny for the gene(s) being transferred, although a backcrossconversion with a stably introgressed trait may also be maintained byfurther backcrossing to the recurrent parent with selection for theconverted trait.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. Specific toalfalfa, see, for example, “Efficient Agrobacterium-mediatedtransformation of alfalfa using secondary somatic embryogenic callus”,Journal of the Korean Society of Grassland Science 20 (1): 13-18 2000,E. Charles Brummer, “Applying Genomics to Alfalfa Breeding Programs”Crop Sci. 44:1904-1907 (2004), and “Genetic transformation of commercialbreeding populations of alfalfa (Medicago sativa)” Plant Cell Tissue andOrgan Culture 42 (2): 129-140 1995 which are incorporated by referencefor this purpose. In addition, expression vectors and in vitro culturemethods for plant cell or tissue transformation and regeneration ofplants are available. See, for example, Gruber et al., “Vectors forPlant Transformation” in Methods in Plant Molecular Biology andBiotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc.,Boca Raton, 1993) pages 89-119.

The most prevalent types of plant transformation involve theconstruction of an expression vector. Such a vector comprises a DNAsequence that contains a gene under the control of or operatively linkedto a regulatory element, for example a promoter. The vector may containone or more genes and one or more regulatory elements.

A genetic trait which has been engineered into the genome of aparticular alfalfa plant using transformation techniques or geneediting, could be moved into the genome of another population usingtraditional breeding techniques that are well known in the plantbreeding arts. For example, a backcrossing approach may be used to movea transgene from a transformed or edited alfalfa plant to an elitepopulation, and the resulting progeny would then comprise thetransgene(s) or edited genes.

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to, genes;coding sequences; inducible, constitutive, and tissue specificpromoters; enhancing sequences; and signal and targeting sequences. Forexample, see the traits, genes and transformation methods listed in U.S.Pat. No. 6,118,055.

Transgenic plants which produce a foreign protein in commercialquantities are provided. For example, techniques for the selection andpropagation of transformed plants, including those well understood inthe art, may yield a plurality of transgenic plants that can beharvested, such as in a conventional manner, and a foreign protein thencan be extracted from a tissue of interest or from total biomass.Methods for protein extraction from plant biomass are provided, such asthose accomplished by methods which are discussed, for example, by Heneyand Orr, Anal. Biochem. 114: 92-6 (1981).

A genetic map can be generated, primarily via conventional RestrictionFragment Length Polymorphisms (RFLP), Polymerase Chain Reaction (PCR)analysis, Simple Sequence Repeats (SSR) and Single NucleotidePolymorphisms (SNP) that identifies the approximate chromosomal locationof the integrated DNA molecule. For exemplary methodologies in thisregard, see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology 269-284 (CRC Press, Boca Raton, 1993). Specific toalfalfa, see Construction of an improved linkage map of diploid alfalfa(Medicago sativa), Theoretical and Applied Genetics 100 (5): 641-657March, 2000 and Isolation of a full-length mitotic cyclin cDNA cloneCyclIIMs from Medicago sativa: Chromosomal mapping and expression, PlantMolecular Biology 27 (6): 1059-1070 1995 which are incorporated byreference for this purpose.

Wang et al. discuss “Large Scale Identification, Mapping and Genotypingof Single-Nucleotide Polymorphisms in the Human Genome”, Science,280:1077-1082, 1998, and similar capabilities are becoming increasinglyavailable for many plant genomes. Map information concerning chromosomallocation is useful for proprietary protection of a subject transgenicplant. If unauthorized propagation is undertaken and crosses made withother germ plasm, the map of the integration region can be compared tosimilar maps for suspect plants to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques. SNPs may also be used alone or in combinationwith other techniques.

Provided are plants genetically engineered to express various phenotypesof agronomic interest and methods for making and using such plants.Through the transformation of alfalfa gene expression can be altered toenhance, for example, disease resistance, insect resistance, herbicideresistance, agronomic properties, grain quality, nutritional quality,digestibility and other traits.

Transgenes and transformation methods facilitate engineering of thegenome of plants to contain and express heterologous genetic elements,such as foreign genetic elements, or additional copies of endogenouselements, or modified versions of native or endogenous genetic elementsin order to alter at least one trait of a plant in a specific manner.Any sequences, such as DNA, whether from a different species or from thesame species, which have been stably inserted into a genome usingtransformation are referred to herein collectively as “transgenes”and/or “transgenic events”. Transgenes can be moved from one genome toanother using breeding techniques which may include, for example,crossing, backcrossing or double haploid production. In someembodiments, a transformed variant of 14XXP21R may comprise at least onetransgene but could contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10and/or no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.Transformed versions of alfalfa variety 14XXP21R containing andinheriting the transgene thereof are provided.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available.

In general, methods to transform, modify, edit or alter plant endogenousgenomic DNA include altering the plant native DNA sequence or apre-existing transgenic sequence including regulatory elements, codingand non-coding sequences. These methods can be used, for example, totarget nucleic acids to pre-engineered target recognition sequences inthe genome. Such pre-engineered target sequences may be introduced bygenome editing or modification. As an example, a genetically modifiedplant variety is generated using “custom” or engineered endonucleasessuch as meganucleases produced to modify plant genomes (see e.g., WO2009/114321; Gao et al. (2010) Plant Journal 1:176-187). Anothersite-directed engineering method is through the use of zinc fingerdomain recognition coupled with the restriction properties ofrestriction enzyme. See e.g., Urnov, et al., (2010) Nat Rev Genet.11(9):636-46; Shukla, et al., (2009) Nature 459 (7245):437-41. Atranscription activator-like (TAL) effector-DNA modifying enzyme (TALEor TALEN) is also used to engineer changes in plant genome. See e.g.,US20110145940, Cermak et al., (2011) Nucleic Acids Res. 39(12) and Bochet al., (2009), Science 326(5959): 1509-12. Site-specific modificationof plant genomes can also be performed using the bacterial type IICRISPR (clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated) system. See e.g., Belhaj et al., (2013), PlantMethods 9: 39; The Cas9/guide RNA-based system allows targeted cleavageof genomic DNA guided by a customizable small noncoding RNA in plants(see e.g., WO 2015026883A1).

Plant transformation methods may involve the construction of anexpression vector. Such a vector comprises a DNA sequence that containsa gene under the control of or operatively linked to a regulatoryelement, for example a promoter. The vector may contain one or moregenes and one or more regulatory elements.

A transgenic event which has been stably engineered into the germ cellline of a particular alfalfa plant using transformation techniques,could be moved into the germ cell line of another variety usingtraditional breeding techniques that are well known in the plantbreeding arts. These varieties can then be crossed to generate analfalfa plant such as alfalfa variety plant 14XXP21R which comprises atransgenic event. For example, a backcrossing approach is commonly usedto move a transgenic event from a transformed alfalfa plant to anothervariety, and the resulting progeny would then comprise the transgenicevent(s). Also, if an inbred variety was used for the transformationthen the transgenic plants could be crossed to a different inbred inorder to produce a transgenic alfalfa plant.

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to, genes;coding sequences; inducible, constitutive, and tissue specificpromoters; enhancing sequences; and signal and targeting sequences. Forexample, see the traits, genes and transformation methods listed in U.S.Pat. Nos. 6,118,055 and 6,284,953. In addition, transformability of avariety can be increased by introgressing the trait of hightransformability from another variety known to have hightransformability, such as Hi-II. See U.S. Patent Application PublicationUS 2004/0016030.

With transgenic or genetically modified plants, a foreign protein can beproduced in commercial quantities. Thus, techniques for the selectionand propagation of transformed plants, which are well understood in theart, yield a plurality of transgenic or genetically modified plants thatare harvested in a conventional manner, and a foreign protein then canbe extracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Sack, M. et al., Curr. Opin. Biotech 32:163-170 (2015).

Transgenic events can be mapped by one of ordinary skill in the art andsuch techniques are well known to those of ordinary skill in the art.

Plants can be genetically engineered or modified to express variousphenotypes of agronomic interest. Through the transformation ormodification of alfalfa the expression of genes can be altered toenhance disease resistance, insect resistance, herbicide tolerance,agronomic traits, grain quality and other traits. Transformation canalso be used to insert DNA sequences which control or help controlmale-sterility. DNA sequences native to alfalfa as well as non-nativeDNA sequences can be transformed into alfalfa and used to alter levelsof native or non-native proteins. Various promoters, targetingsequences, enhancing sequences, and other DNA sequences can be insertedinto the alfalfa genome for the purpose of altering the expression ofproteins. Reduction of the activity of specific genes (also known asgene silencing, or gene suppression) is desirable for several aspects ofgenetic engineering in plants.

Many techniques for gene silencing are well known to one of skill in theart, including but not limited to knock-outs (such as by insertion of atransposable element such as mu or other genetic elements such as a FRT,Lox or other site specific integration site, antisense technology (see,e.g., U.S. Pat. Nos. 5,107,065; 5,453,566; and 5,759,829);co-suppression (e.g., U.S. Pat. No. 5,034,323), virus-induced genesilencing; target-RNA-specific ribozymes; hairpin structures (WO99/53050 and WO 98/53083); MicroRNA; ribozymes; oligonucleotide mediatedtargeted modification (e.g., WO 03/076574 and WO 99/25853); Zn-fingertargeted molecules (e.g., WO 01/52620; WO 03/048345; and WO 00/42219);and other methods or combinations of the above methods known to those ofskill in the art.

Exemplary nucleotide sequences that may be altered by geneticengineering include, but are not limited to, those categorized below.Exemplary nucleotide sequences that may be altered by geneticengineering include, but are not limited to, those categorized below.

1. Transgenes That Confer Resistance to Insects or Disease and ThatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. A plant resistant to a disease is one that is moreresistant to a pathogen as compared to the wild type plant.

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. DNA molecules encodingdelta-endotoxin genes can be purchased from American Type CultureCollection (Manassas, Va.), for example, under ATCC Accession Nos.40098, 67136, 31995 and 31998. Other non-limiting examples of Bacillusthuringiensis transgenes being genetically engineered are given in thefollowing patents and patent applications: U.S. Pat. Nos. 5,188,960;5,689,052; 5,880,275; 5,986,177; 7,105,332; 7,208,474; WO 91/14778; WO99/31248; WO 01/12731; WO 99/24581; WO 97/40162 and U.S. applicationSer. Nos. 10/032,717; 10/414,637; 11/018,615; 11/404,297; 11/404,638;11/471,878; 11/780,501; 11/780,511; 11/780,503; 11/953,648; and Ser. No.11/957,893.

(C) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof.

(D) An insect-specific peptide which, upon expression, disrupts thephysiology of the affected pest. For example, an insect diuretic hormonereceptor or an allostatin. See also U.S. Pat. No. 5,266,317 disclosinggenes encoding insect-specific toxins.

(E) An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(F) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also U.S. Pat. Nos. 6,563,020;7,145,060 and 7,087,810.

(G) A molecule that stimulates signal transduction. For example,calmodulin cDNA clones.

(H) A hydrophobic moment peptide. See PCT application WO 95/16776 andU.S. Pat. No. 5,580,852 disclosure of peptide derivatives of Tachyplesinwhich inhibit fungal plant pathogens) and PCT application WO 95/18855and U.S. Pat. No. 5,607,914 (teaches synthetic antimicrobial peptidesthat confer disease resistance).

(I) A membrane permease, a channel former or a channel blocker.

(J) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. Coat protein-mediated resistance may beenconferred upon transformed plants against, for example, alfalfa mosaicvirus, cucumber mosaic virus, tobacco streak virus, potato virus X,potato virus Y, tobacco etch virus, tobacco rattle virus and tobaccomosaic virus.

(K) An insect-specific antibody or an immunotoxin derived therefrom. Forexample, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect.

(L) A virus-specific antibody. Plants expressing recombinant antibodygenes may be protected from virus attack.

(M) A developmental-arrestive protein produced in nature by a pathogenor a parasite. For example, fungal endo alpha-1,4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-alpha-1,4-D-galacturonase.

(N) A developmental-arrestive protein produced in nature by a plant. Forexample, plants expressing the barley ribosome-inactivating gene mayhave an increased resistance to fungal disease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes

(P) Antifungal genes. See, e.g., U.S. application Ser. Nos. 09/950,933;11/619,645; 11/657,710; 11/748,994; 11/774,121 and U.S. Pat. Nos.6,891,085 and 7,306,946.

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see U.S. Pat. Nos. 5,716,820; 5,792,931; 5,798,255;5,846,812; 6,083,736; 6,538,177; 6,388,171 and 6,812,380.

(R) Cystatin and cysteine proteinase inhibitors. See U.S. Pat. No.7,205,453.

(S) Defensin genes. See, e.g., WO03000863 and U.S. Pat. Nos. 6,911,577;6,855,865; 6,777,592 and 7,238,781.

(T) Genes conferring resistance to nematodes. See, e.g., PCT ApplicationWO96/30517; PCT Application WO93/19181, WO 03/033651 and U.S. Pat. Nos.6,284,948 and 7,301,069.

(U) Genes that confer resistance to Phytophthora Root Rot, such as theRps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes.

(V) Genes that confer resistance to Brown Stem Rot, such as described inU.S. Pat. No. 5,689,035.

(W) Genes that confer resistance to Colletotrichum, such as described inUS Patent publication US20090035765. This includes the Rcg locus thatmay be utilized as a single locus conversion.

2. Transgenes That Confer Tolerance to A Herbicide, For Example:

(A) A herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant acetolactate synthase (ALS) and acetohydroxyacid synthase(AHAS) enzyme as described, for example, in U.S. Pat. Nos. 5,605,011;5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373;5,331,107; 5,928,937; and 5,378,824; US Patent Publication No.20070214515, and international publication WO 96/33270.

(B) Glyphosate (tolerance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes), andpyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835,which discloses the nucleotide sequence of a form of EPSPS which canconfer glyphosate tolerance. U.S. Pat. No. 5,627,061 also describesgenes encoding EPSPS enzymes. See also U.S. Pat. Nos. 6,566,587;6,338,961; 6,248,876 B1; 6,040,497; 5,804,425; 5,633,435; 5,145,783;4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114 B1;6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re.36,449; RE 37,287 E; and 5,491,288; and international publicationsEP1173580; WO 01/66704; EP1173581 and EP1173582.

Glyphosate tolerance is also imparted to plants that express a gene thatencodes a glyphosate oxido-reductase enzyme as described more fully inU.S. Pat. Nos. 5,776,760 and 5,463,175. In addition, glyphosatetolerance can be imparted to plants by the over expression of genesencoding glyphosate N-acetyltransferase. See, for example,US2004/0082770; US2005/0246798; and US2008/0234130. A DNA moleculeencoding a mutant aroA gene can be obtained under ATCC accession No.39256, and the nucleotide sequence of the mutant gene is disclosed inU.S. Pat. No. 4,769,061. European Patent Application No. 0 333 033 andU.S. Pat. No. 4,975,374 disclose nucleotide sequences of glutaminesynthetase genes which confer tolerance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in European PatentNos. 0 242 246 and 0 242 236. See also, U.S. Pat. Nos. 5,969,213;5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477;5,646,024; 6,177,616 B1; and 5,879,903. Exemplary genes conferringresistance to phenoxy propionic acids, cyclohexanediones andcyclohexones, such as sethoxydim and haloxyfop, are the Acc1-S1, Acc1-S2and Acc1-S3 genes.

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes), glutathione S-transferase and a benzonitrile (nitrilasegene) such as bromoxynil. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648 to Stalker, and DNA moleculescontaining these genes are available under ATCC Accession Nos. 53435,67441 and 67442.

(D) Other genes that confer tolerance to herbicides include: a geneencoding a chimeric protein of rat cytochrome P4507A1 and yeastNADPH-cytochrome P450 oxidoreductase, genes for glutathione reductaseand superoxide dismutase, and genes for various phosphotransferases.

(E) A herbicide that inhibits protoporphyrinogen oxidase (protox or PPO)is necessary for the production of chlorophyll, which is necessary forall plant survival. The protox enzyme serves as the target for a varietyof herbicidal compounds. PPO-inbibitor herbicides can inhibit growth ofall the different species of plants present, causing their totaldestruction. The development of plants containing altered protoxactivity which are tolerant to these herbicides are described, forexample, in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1; and 5,767,373;and international patent publication WO 01/12825.

(F) Dicamba (3,6-dichloro-2-methoxybenzoic acid) is an organochloridederivative of benzoic acid which functions by increasing plant growthrate such that the plant dies.

3. Transgenes That Confer or Contribute to an Altered GrainCharacteristic, Such as:

(A) Altered fatty acids, for example, by

(1) Down-regulation of stearoyl-ACP desaturase to increase stearic acidcontent of the plant. See, e.g., WO99/64579,

(2) Elevating oleic acid via FAD-2 gene modification and/or decreasinglinolenic acid via FAD-3 gene modification (se, e.g., U.S. Pat. Nos.6,063,947; 6,323,392; 6,372,965 and WO 93/11245),

(3) Altering conjugated linolenic or linoleic acid content, such as inWO 01/12800,

(4) Altering LEC1, AGP, Dek1, Superal1, mi1ps, various Ipa genes such asIpa1, Ipa3, hpt or hggt. For example, see WO 02/42424, WO 98/22604, WO03/011015, WO02/057439, WO03/011015, U.S. Pat. Nos. 6,423,886,6,197,561, 6,825,397, and U.S. Application Serial Nos. US2003/0079247,US2003/0204870.

(B) Altered phosphate content, for example, by the

(1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant.

(2) Modulating a gene that reduces phytate content. In alfalfa, this,for example, could be accomplished, by cloning and then re-introducingDNA associated with one or more of the alleles, such as the LPA alleles,identified in alfalfa mutants characterized by low levels of phyticacid.

(C) Altered carbohydrates affected, for example, by altering a gene foran enzyme that affects the branching pattern of starch or, a genealtering thioredoxin such as NTR and/or TRX (See U.S. Pat. No.6,531,648) and/or a gamma zein knock out or mutant such as cs27 orTUSC27 or en27 (See U.S. Pat. No. 6,858,778 and US2005/0160488,US2005/0204418). See e.g., WO 99/10498 (improved digestibility and/orstarch extraction through modification of UDP-D-xylose 4-epimerase,Fragile 1 and 2, Ref1, HCHL, C4H) and U.S. Pat. No. 6,232,529 (method ofproducing high oil seed by modification of starch levels (AGP)). Thefatty acid modification genes mentioned herein may also be used toaffect starch content and/or composition through the interrelationshipof the starch and oil pathways.

(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see U.S. Pat. No. 6,787,683,US2004/0034886 and WO 00/68393 involving the manipulation of antioxidantlevels, and WO 03/082899 through alteration of a homogentisate geranyltransferase (hggt).

(E) Altered essential seed amino acids. For example, see U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds), U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389(high lysine), WO99/40209 (alteration of amino acid compositions inseeds), WO99/29882 (methods for altering amino acid content ofproteins), U.S. Pat. No. 5,850,016 (alteration of amino acidcompositions in seeds), WO98/20133 (proteins with enhanced levels ofessential amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S.Pat. No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plantamino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increasedlysine and threonine), U.S. Pat. No. 6,441,274 (plant tryptophansynthase beta subunit), U.S. Pat. No. 6,346,403 (methionine metabolicenzymes), U.S. Pat. No. 5,939,599 (high sulfur), U.S. Pat. No. 5,912,414(increased methionine), WO98/56935 (plant amino acid biosyntheticenzymes), WO98/45458 (engineered seed protein having higher percentageof essential amino acids), WO98/42831 (increased lysine), U.S. Pat. No.5,633,436 (increasing sulfur amino acid content), U.S. Pat. No.5,559,223 (synthetic storage proteins with defined structure containingprogrammable levels of essential amino acids for improvement of thenutritional value of plants), WO96/01905 (increased threonine),WO95/15392 (increased lysine), US2003/0163838, US2003/0150014,US2004/0068767, U.S. Pat. No. 6,803,498, WO01/79516.

4. Genes that Control Male-sterility: There are several methods ofconferring genetic male sterility available, such as multiple mutantgenes at separate locations within the genome that confer malesterility, as disclosed in U.S. Pat. Nos. 4,654,465 and 4,727,219 toBrar et al. and chromosomal translocations as described by Patterson inU.S. Pat. Nos. 3,861,709 and 3,710,511. In addition to these methods,Albertsen et al., U.S. Pat. No. 5,432,068, describe a system of nuclearmale sterility which includes: identifying a gene which is needed formale fertility; silencing this native gene which is needed for malefertility; removing the native promoter from the essential malefertility gene and replacing it with an inducible promoter; insertingthis genetically engineered gene back into the plant; and thus creatinga plant that is male sterile because the inducible promoter is not “on”resulting in the male fertility gene not being transcribed. Fertility isrestored by inducing, or turning “on”, the promoter, which in turnallows the gene that confers male fertility to be transcribed.

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT (WO 01/29237).

(B) Introduction of various stamen-specific promoters (WO 92/13956, WO92/13957).

(C) Introduction of the barnase and the barstar gene.

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341; 6,297,426; 5,478,369;5,824,524; 5,850,014; and 6,265,640.

5. Genes that create a site for site specific DNA integration. Thisincludes the introduction of FRT sites that may be used in the FLP/FRTsystem and/or Lox sites that may be used in the Cre/Loxp system. Forexample, see WO 99/25821. Other systems that may be used include the Ginrecombinase of phage Mu, the Pin recombinase of E. coli, and the R/RSsystem of the pSR1 plasmid.

6. Genes that affect abiotic stress resistance (including but notlimited to flowering, ear and seed development, enhancement of nitrogenutilization efficiency, altered nitrogen responsiveness, droughtresistance or tolerance, cold resistance or tolerance, and saltresistance or tolerance) and increased yield under stress. For example,see: WO 00/73475 where water use efficiency is altered throughalteration of malate; U.S. Pat. Nos. 5,892,009; 5,965,705; 5,929,305;5,891,859; 6,417,428; 6,664,446; 6,706,866; 6,717,034; 6,801,104;WO2000060089; WO2001026459; WO2001035725; WO2001034726; WO2001035727;WO2001036444; WO2001036597; WO2001036598; WO2002015675; WO2002017430;WO2002077185; WO2002079403; WO2003013227; WO2003013228; WO2003014327;WO2004031349; WO2004076638; WO9809521; and WO9938977 describing genes,including CBF genes and transcription factors effective in mitigatingthe negative effects of freezing, high salinity, and drought on plants,as well as conferring other positive effects on plant phenotype;US2004/0148654 and WO01/36596 where abscisic acid is altered in plantsresulting in improved plant phenotype such as increased yield and/orincreased tolerance to abiotic stress; WO2000/006341, WO04/090143, U.S.application Ser. Nos. 10/817,483 and 09/545,334 where cytokininexpression is modified resulting in plants with increased stresstolerance, such as drought tolerance, and/or increased yield. Also seeWO0202776, WO2003052063, JP2002281975, U.S. Pat. No. 6,084,153,WO0164898, U.S. Pat. Nos. 6,177,275, and 6,107,547 (enhancement ofnitrogen utilization and altered nitrogen responsiveness). For ethylenealteration, see US20040128719, US20030166197 and WO200032761. For planttranscription factors or transcriptional regulators of abiotic stress,see e.g. US20040098764 or US20040078852.

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see e.g.WO97/49811 (LHY), WO98/56918 (ESD4), WO97/10339 and U.S. Pat. No.6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO96/14414 (CON),WO96/38560, WO01/21822 (VRN1), WO00/44918 (VRN2), WO99/49064 (GI),WO00/46358 (FRI), WO97/29123, U.S. Pat. Nos. 6,794,560, 6,307,126 (GAI),WO99/09174 (D8 and Rht), WO2004076638 and WO2004031349 (transcriptionfactors).

Seed Treatments and Cleaning

Methods of harvesting the seeds of variety 14XXP21R and using the seedsfor planting are provided. Also provided are methods of using the seedof variety 14XXP21R, or grain harvested from variety 14XXP21R, as seedfor planting. Embodiments include cleaning the seed, treating the seed,and/or conditioning the seed and seed produced by such cleaning,conditioning, treating or any combination thereof. Cleaning the seed isunderstood in the art to include removal of one or more of foreigndebris such as weed seed, chaff, and non-seed plant matter from theseed. Conditioning the seed is understood in the art to includecontrolling the temperature and rate of dry down of the seed and storingthe seed in a controlled temperature environment. Seed treatment is theapplication of a composition to the seed such as a coating or powder.Methods for producing a treated seed include the step of applying acomposition to the seed or seed surface. Seeds are provided which haveon the surface a composition. Biological active components such asbacteria can also be used as a seed treatment. Some examples ofcompositions include active components such as insecticides, fungicides,pesticides, antimicrobials, germination inhibitors, germinationpromoters, cytokinins, and nutrients. Biological active components, suchas bacteria, can also be used as a seed treatment. Carriers such aspolymers can be used to increase binding of the active component to theseed.

To protect and to enhance yield production and trait technologies, seedtreatment options can provide additional crop plan flexibility and costeffective control against insects, weeds and diseases, thereby furtherenhancing the invention described herein. Seed material can be treated,typically surface treated, with a composition comprising combinations ofchemical or biological herbicides, herbicide safeners, insecticides,fungicides, germination inhibitors and enhancers, nutrients, plantgrowth regulators and activators, bactericides, nematicides, avicidesand/or molluscicides. These compounds are typically formulated togetherwith further carriers, surfactants or application-promoting adjuvantscustomarily employed in the art of formulation. The coatings may beapplied by impregnating propagation material with a liquid formulationor by coating with a combined wet or dry formulation. Examples of thevarious types of compounds that may be used as seed treatments areprovided in The Pesticide Manual: A World Compendium, C. D. S. TomlinEd., Published by the British Crop Production Council.

Some seed treatments that may be used on crop seed include, but are notlimited to, one or more of abscisic acid, acibenzolar-S-methyl,avermectin, amitrol, azaconazole, azospirillum, azadirachtin,azoxystrobin, Bacillus spp. (including one or more of cereus, firmus,megaterium, pumilis, sphaericus, subtilis and/or thuringiensis),Bradyrhizobium spp. (including one or more of betae, canariense,elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/oryuanmingense), captan, carboxin, chitosan, clothianidin, copper,cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil,fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein,imazalil, imidacloprid, ipconazole, isoflavenoids,lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam,metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium,penthiopyrad, permethrine, picoxystrobin, prothioconazole,pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB,tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram,tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin,triticonazole and/or zinc. PCNB seed coat refers to EPA registrationnumber 00293500419, containing quintozen and terrazole. TCMTB refers to2-(thiocyanomethylthio) benzothiazole.

Seed varieties and seeds with specific transgenic traits may be testedto determine which seed treatment options and application rates maycomplement such varieties and transgenic traits in order to enhanceyield. For example, a variety with good yield potential but head smutsusceptibility may benefit from the use of a seed treatment thatprovides protection against head smut, a variety with good yieldpotential but cyst nematode susceptibility may benefit from the use of aseed treatment that provides protection against cyst nematode, and soon. Likewise, a variety encompassing a transgenic trait conferringinsect resistance may benefit from the second mode of action conferredby the seed treatment, a variety encompassing a transgenic traitconferring herbicide resistance may benefit from a seed treatment with asafener that enhances the plants resistance to that herbicide, etc.Further, the good root establishment and early emergence that resultsfrom the proper use of a seed treatment may result in more efficientnitrogen use, a better ability to withstand drought and an overallincrease in yield potential of a variety or varieties containing acertain trait when combined with a seed treatment.

Industrial Applicability

Another embodiment is a method of harvesting the grain or plant materialof the variety 14XXP21R and using the grain or plant material in acommodity, such as used for forage. Commodity products may contain atleast one cell of alfalfa variety 14XXP21R or at least one cell of amodified plant of the variety disclosed herein. Methods of producing aplant product or commodity from the alfalfa variety disclosed herein arealso provided. Examples of alfalfa grain or plant material as acommodity plant product include, but are not limited to, hay, haylage,forage, sprouts, meal, cellulose, greenchop, and silage, which can beused as livestock feed. Hay, meal and silage from the alfalfa describedherein are provided and their use as livestock feed or bedding, forexample for horses, beef cattle, dairy cattle, hogs, sheep, poultry,chickens, turkeys and other farm animals as well as in the pet industrysuch as for rodents and reptiles. The food and nutritional uses ofalfalfa include alfalfa sprouts for human consumption and nutritionalsupplements.

Processing the seed or grain can include one or more of cleaning toremove foreign material and debris from the seed or grain, conditioning,such as addition of moisture to the grain, steeping the grain, wetmilling, dry milling and sifting.

The seed of the alfalfa variety, the plant produced from the seed, aplant produced from crossing of alfalfa variety 14XXP21R and variousparts of the alfalfa plant and transgenic and locus converted versionsof the foregoing, can be utilized for human food, livestock feed, and asa raw material in industry.

All publications, patents, and patent applications mentioned in thespecification are incorporated by reference herein for the purpose citedto the same extent as if each was specifically and individuallyindicated to be incorporated by reference herein.

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding. Asis readily apparent to one skilled in the art, the foregoing are onlysome of the methods and compositions that illustrate the embodiments ofthe foregoing invention. It will be apparent to those of ordinary skillin the art that variations, changes, modifications, and alterations maybe applied to the compositions and/or methods described herein withoutdeparting from the true spirit, concept, and scope of the invention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains”, “containing,” “characterizedby” or any other variation thereof, are intended to cover anon-exclusive inclusion.

Unless expressly stated to the contrary, “or” is used as an inclusiveterm. For example, a condition A or B is satisfied by any one of thefollowing: A is true (or present) and B is false (or not present), A isfalse (or not present) and B is true (or present), and both A and B aretrue (or present). The indefinite articles “a” and “an” preceding anelement or component are nonrestrictive regarding the number ofinstances (i.e., occurrences) of the element or component. Therefore “a”or “an” should be read to include one or at least one, and the singularword form of the element or component also includes the plural unlessthe number is obviously meant to be singular.

DEPOSITS

Applicant has made a deposit of at least 625 seeds of alfalfa variety14XXP21R with the Provasoli-Guillard National Center for Marine Algaeand Microbiota (NCMA), 60 Bigelow Drive, East Boothbay, Me. 04544, USA,with NCMA Accession Number 202112010, which has been accepted under theBudapest Treaty. The seeds deposited with the NCMA on Dec. 10, 2021 wereobtained from the seed of the variety maintained by Pioneer Hi-BredInternational, Inc., 7250 NW 62nd Avenue, Johnston, Iowa, 50131 sinceprior to the filing date of this application. Access to this seed willbe available during the pendency of the application to the Commissionerof Patents and Trademarks and persons determined by the Commissioner tobe entitled thereto upon request. Upon allowance of any claims in theapplication, the Applicant will make the deposit available to the publicpursuant to 37 C.F.R. § 1.808. This deposit of the Alfalfa Variety14XXP21R will be maintained in the NCMA depository, which is a publicdepository, for a period of 30 years, or 5 years after the most recentrequest, or for the enforceable life of the patent, whichever is longer,and will be replaced if it becomes nonviable during that period.Additionally, Applicant has or will satisfy all of the requirements of37 C.F.R. §§ 1.801-1.809, including providing an indication of theviability of the sample upon deposit. Applicant has no authority towaive any restrictions imposed by law on the transfer of biologicalmaterial or its transportation in commerce. Applicant does not waive anyinfringement of rights granted under this patent or under the PlantVariety Protection Act (7 USC 2321 et seq.).

Example 1: Breeding History

Alfalfa variety 14XXP21R is an intracross of parent plants selected fromproprietary non-public experimental lines high in alfalfa stem nematoderesistance, forage yield, persistence, forage quality, standability,high resistance to Aphanomyces root rot (Race 2), and/or resistance toone or more of the following diseases and pests: bacterial wilt,Fusarium wilt, Verticillium wilt, Phytophthora root rot, and Aphanomycesroot rot (Race 1). Parent plants were identified using phenotypicselection in selection nurseries for standability (lodging resistance),forage quality, persistence, agronomic characteristics, and improvedforage yield. Parent plants contain tolerance to glyphosate herbicideconferred by the CP4 5-enolpyruvylshikimate-3-phosphate synthase(cp4-epsps) transgene, MON-00101.

Example 2: Variety Description

Alfalfa variety 14XXP21R is a moderately dormant variety with falldormancy similar to FD class 4 check varieties. Flower color isapproximately 74% purple, 25% variegated with traces of cream, white,and yellow. Alfalfa variety 14XXP21R is very winterhardy and is tolerantto glyphosate herbicide. The variety is highly resistant to Anthracnose(Race 1), bacterial wilt, Aphanomyces root rot (Race 1), Aphanomycesroot rot (Race 2), Phytophthora root rot, Fusarium wilt, Verticilliumwilt, and stem nematode. It is resistant to pea aphid and spottedalfalfa aphid.

The characteristics of alfalfa variety 14XXP21R are described in thefollowing tables.

TABLE 1 Yield data for 14XXP21R in DM in T/A compared to other varietiesat multiple locations. Test Syn No. WL Location Gen cuts 14XXP21R 365HQ54Q14 FSG 329 LSD 0.05 CV % Connell, 2 5 12.1 11.7 11.8 11.4 1.5 14.1 WAMoses 2 4 11.9 13 11.2 11.6 1 4.9 Lake, WA Rosemont, 2 4 5.8 5.7 5.7 5.80.5 5 MN Landisville, 2 4 10.3 9 9.8 9.7 0.9 5.3 PA Arlington, 2 4 10.610.1 8.8 8.6 1.1 7.1 WI Arlington, 2 4 9.9 8.8 9.2 8.8 0.7 4.6 WISlinger, WI 2 4 6.5 6.3 6.3 6.2 0.9 8.9

TABLE 2 Mean Annual Yield in Tons DM/Acre. # of Total # Variety Years ofWL FSG names Harvested Harvests 14XXP21R 365HQ 54Q14 329 14XXP21R 7 299.6 Check 1 7 29 9.6 9.2 (WL 365HQ) Check 2 7 29 9.6 9.0 (54Q14) Check 37 29 9.6 8.9 (FSG 329)

TABLE 3 Persistence data for 14XXP21R compared to other varieties(Percent of stand). % Stand Check Varieties Syn No. of Years No. of14XXP21R L-504HD LegenDairy XHD LSD Gen Harvested Harvests Initial/FinalInitial/Final Initial/Final .05 CV % 2 2 8 100/99 100/95 100/93 .2/7.1.1/4.9

TABLE 4 Fall dormancy as determined from spaced plantings Fall DormancySyn Unadjusted Variety Class Gen FDR 14XXP21R* 2 12.9 Maverick  1 Vernal 2 5246  3 11.6 Legend  4 12.9 Archer  5 14.2 ABI 700  6 Dona Ana  7Pierce  8 CUF 101  9 UC-1887 10 UC-1465 11 Test Mean: 12.7L.S.D._((0.05%))  1.8 C.V. (%) 10.4 *14XXP21R is in fall dormancy class4 used by the NAFA. (4 = Dormant to Moderately-Dormant)

TABLE 5 Winterhardiness for 14XXP21R Check Class Syn 1 2 3 4 5 6 Gen14XXP21R* ZG 9830 5262 WL325 HQ G-2852 Archer CUF 101 LSD 0.05 CV % 12.6 2.1 2.3 2.9 3.7 4.0 5.0 0.6 14.7 2 2.6 2.5 2.7 2.9 3.7 3.9 4.9 0.615.5 *14XXP21R is most similar to winter survival class 2, verywinterhardy

TABLE 6 Anthracnose (Race 1) Disease Scores for 14XXP21R - greenhousetest. Resistance Syn Unadjusted Adjusted Variety Class Gen % R % R Score14XXP21R HR 2 62 72 Arc HR 59 70 Saranac AR R Saranac S  2  2 Test Mean:46 55 L.S.D. (.05%)  9 11 C.V. (%) 14 14

TABLE 7 Bacterial Wilt Disease Scores for 14XXP21R - greenhouse testResistance Syn Unadjusted Adjusted Variety Class Gen % R % R Score14XXP21R HR 2 45 60 Vernal R 30 40 Narragansett S Sonora S  4  5 TestMean: 43 57 L.S.D. (.05%)  9 12 C.V. (%) 16 16

TABLE 8 Fusarium Wilt Disease Scores for 14XXP21R - greenhouse test.Resistance Syn Unadjusted Adjusted Variety Class Gen % R % R Score14XXP21R HR 2 53 67 Agate HR 36 45 Moapa 69 HR Narragansett MR MNGN-1 S 3  4 Test Mean: 51 65 L.S.D. (.05%)  8 10 C.V. (%) 13 13

TABLE 9 Verticillium Wilt Disease Scores for 14XXP21R - greenhouse testResistance Syn Unadjusted Adjusted Variety Class Gen % R % R Score14XXP21R HR 2 43 54 Vertus R 32 40 Oneida VR HR Saranac S  4  5 TestMean: 39 48 L.S.D. (.05%) 11 14 C.V. (%) 21 21

TABLE 10 Phytophthora Root Rot Disease Scores for 14XXP21R - seedlingand greenhouse test. Resistance Syn Unadjusted Adjusted Variety ClassGen % R % R Score 14XXP21R HR 2 76 77 WAPH-1 HR 54 55 (seedling) MNP-D1R (seedling) Agate R Saranac S  3  3 Test Mean: 63 64 L.S.D. (.05%) 1313 C.V. (%) 15 15

TABLE 11 Aphanomyces Root Rot Disease Scores for 14XXP21R - greenhousetest - Race 1. Resistance Syn Unadjusted Adjusted Variety Class Gen % R% R Score 14XXP21R HR 2 74 80 WAPH-1 (Race 1) R 47 50 WAPH-1 (Race 2) SWAPH-5 (Race 2) R Saranac (Races 1 S  4  4 & 2) Test Mean: 60 64 L.S.D.(.05%) 15 16 C.V. (%) 18 18

TABLE 12 Pea Aphid Insect Scores for Scores for 14XXP21R - greenhousetest. Resistance Syn Unadjusted Adjusted Variety Class Gen % R % R Score14XXP21R R 2 30 37 CUF-101 HR PA-1 HR Kanza R Baker R 36 45 Caliverde SMoapa 69 S Vernal S  2  3 Ranger S Test Mean: 37 46 L.S.D. 11 14 (.05%)C.V. (%) 22 22

TABLE 13 Spotted Alfalfa Aphid Insect Scores for 14XXP21R - greenhousetest. Resistance Syn Unadjusted Adjusted Variety Class Gen % R % R Score14XXP21R R 2 34 41 CUF-101 HR 50 60 Baker R Mesa Sirsa R Kanza RCaliverde S Arc S OK08 S Ranger S  3  4 Test Mean: 34 42 L.S.D. (.05%)11 13 C.V. (%) 23 23

TABLE 14 Stem Nematode Scores for 14XXP21R - greenhouse test. ResistanceSyn Unadjusted Adjusted Variety Class Gen % R % R Score 14XXP21R HR 2 4454 Vernema HR 49 60 Lahontan R Lew R Ranger S  3  4 Moapa 69 S TestMean: 32 39 L.S.D. (.05%) 11 13 C.V. (%) 25 25

TABLE 15 Aphanomyces Root Rot Disease Scores for 14XXP21R - greenhousetest - Race 2. Resistance Syn Unadjusted Adjusted Variety Class Gen % R% R Score 14XXP21R HR 2 59 72 WAPH-1 (Race 1) R WAPH-1 (Race 2) S  2  3WAPH-5 (Race 2) R 41 50 Saranac (Races 1 S  2  2 & 2) Test Mean: 31 38L.S.D. (.05%) 12 15 C.V. (%) 28 28

All disease tests conducted for National Alfalfa and MiscellaneousLegume Variety Review Board for AOSCA certification and were conductedby standard procedures and scoring systems as described in the NAAICStandard Tests to Characterize Alfalfa Cultivars, maintained online onthe NAAIC website.

We claim:
 1. A seed of alfalfa variety 14XXP21R, representative seedhaving been deposited under NCMA Accession Number
 202112010. 2. Analfalfa plant, or a plant part thereof, produced by growing the seed ofclaim 1, wherein the plant part comprises at least one cell of alfalfavariety 14XXP21R.
 3. A pollen grain or ovule of the plant of claim
 2. 4.A tissue culture of regenerable cells or regenerable protoplasts fromthe plant of claim 2, wherein the tissue culture comprises somaticcells.
 5. The tissue culture according to claim 4, wherein a cell orprotoplast of the tissue culture is derived from a tissue or cellselected from the group consisting of leaves, roots, root tips, roothairs, anthers, pistils, stamens, pollen, ovules, flowers, seeds, stems,buds, cotyledons, hypocotyls, cells and protoplasts.
 6. An alfalfa plantregenerated from the tissue culture of claim 4, wherein the regeneratedplant has all of the morphological and physiological characteristics ofalfalfa variety 14XXP21R, representative seed of said alfalfa varietyhaving been deposited under NCMA Accession Number
 202112010. 7. A methodfor producing a first generation progeny alfalfa seed comprisingcrossing the plant of claim 2 with a second alfalfa plant orself-pollinating the plant of claim 2 and harvesting the resultantalfalfa seed.
 8. A method for producing an alfalfa plant, the methodcomprising introducing a locus conversion or a transgene into the plantor plant part of claim
 2. 9. An alfalfa plant produced by the method ofclaim 8; wherein the alfalfa plant produced comprises the locusconversion or transgene and otherwise comprises all of the physiologicaland morphological characteristics of a plant of the alfalfa variety14XXP21R.
 10. A seed that produces the plant of claim 9, wherein theseed comprises the locus conversion or transgene and produces a plantthat otherwise comprises all of the physiological and morphologicalcharacteristics of a seed of the alfalfa variety 14XXP21R.
 11. The plantof claim 9, wherein the transgene or locus conversion confers a traitselected from the group consisting of herbicide resistance, insectresistance, disease resistance, improved digestibility, improved energycontent, male sterility, and improved winterhardiness.
 12. A method forintroducing a transgene or a single locus conversion into a populationof alfalfa plants, the method comprising the steps of: (a) modifying theplant or plant part of claim 2 by introducing a transgene or a singlelocus conversion; and (b) crossing the modified alfalfa plant or plantpart of step (a) with a population of alfalfa plants to produce apopulation of progeny plants, wherein at least one progeny plantcomprises the transgene or single locus conversion.
 13. A method forproducing a synthetic alfalfa variety, the method comprising crossing aplant grown from the seed of claim 1 with a plant grown from seed of oneor more different alfalfa plants.
 14. A method for producing alfalfaseed, the method comprising growing the plant of claim 2 and allowingthe plant to cross pollinate with one or more different alfalfa plants.15. A method of producing a commodity plant product, the methodcomprising producing the commodity plant product from the plant of claim2, wherein the commodity plant product is selected from the groupconsisting of forage, hay, meal, greenchop, and silage.
 16. A commodityplant product produced by the method of claim 15, wherein the commodityplant product comprises at least one cell of said alfalfa variety14XXP21R.
 17. The seed of claim 1, further comprising a seed treatmenton the surface of the seed.
 18. A method comprising cleaning the seed ofclaim
 1. 19. The method of claim 12, further comprising the step of: (c)applying a selection technique to the population produced in (b) toselect the progeny plants that comprise the transgene or single locusconversion.
 20. A method for producing nucleic acids, the methodcomprising isolating nucleic acids from the seed of claim 1.